氧化苦参碱对氧糖剥夺/复糖复氧损伤星形胶质细胞AMPK/mTOR途径及自噬的影响  

Oxymatrine regulates autophagy through AMPK/mTOR pathway to inhibit OGD/R-induced damage in astrocytes

作　　者：崔明宇 卢金莹 于露 王宇彤 边疆 王高 杨新霞 杨菁 CUI Ming-yu;LU Jin-ying;YU Lu;WANG Yu-tong;BIAN Jiang;WANG Gao;YANG Xin-xia;YANG Jing(Dept of Biochemistry and Molecular Biology,Basic Medical College,Jinzhou Medical University,Jinzhou Liaoning 121001,China)

机构地区：[1]锦州医科大学基础医学院生物化学与分子生物学教研室,辽宁锦州121001

出　　处：《中国药理学通报》2023年第12期2331-2338,共8页Chinese Pharmacological Bulletin

基　　金：辽宁省教育厅面上项目(No 2021LJKZ0823);锦州医科大学一流学科建设项目(No 202203L207)。

摘　　要：目的探讨AMPK/mTOR通路调节的自噬在氧化苦参碱(oxymatrine,OMT)抑制氧糖剥夺/复糖复氧(oxygen-glucose deprivation and reperfusion injury,OGD/R)对星形胶质细胞(astrocyte,AS)损伤中的作用。方法将分离纯化AS随机分为对照组(CON组)、模型组(OGD/R组)和OGD/R+OMT组(0.1、0.2、0.4 mmol·L^(-1))。MTT法检测细胞存活率;Hoechst 33342和Annexin V-FITC法检测细胞凋亡;流式细胞仪检测细胞活性氧(reactive oxygen species,ROS)含量;Western blot法检测p-AMPK、AMPK、p-mTOR、mTOR、Beclin 1、LC3B、p62和β-actin的表达。同时通过加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)探究自噬在OMT药效中作用。结果与CON组相比,OGD/R组细胞存活率明显降低(P<0.01),细胞凋亡率及细胞内ROS生成明显增加(P<0.01),p-AMPK/AMPK和LC3BⅡ/Ⅰ的比值及Beclin 1含量升高(P<0.01),p-mTOR/mTOR比值及p62含量降低(P<0.01)。与OGD/R组相比,OGD/R+OMT组细胞存活率明显升高(P<0.01),细胞凋亡率减少(P<0.01),细胞内ROS生成明显减低(P<0.01),p-AMPK/AMPK和LC3BⅡ/Ⅰ的比值降低(P<0.01),p-mTOR/mTOR比值及p62含量升高(P<0.01)。与OGD/R组相比,OGD/R+3-MA组细胞存活率明显提高(P<0.01),同时OGD/R+3-MA组存活率与OGD/R+OMT及OGD/R+OMT+3-MA组相比均有明显差异(P<0.01)。结论OMT对OGD/R诱导的AS具有保护作用,其中作用与抑制AMPK/mTOR信号通路及改善自噬有关。Aim To investigate the role of autophagy regulated by the AMPK/mTOR pathway in the prevention of oxygen-glucose deprivation/reperfusion injury(OGD/R)in astrocytes using oxymatrine(OMT).Methods The isolated and purified astrocytes(AS)were randomly divided into control group(CON group),OGD/R group and OGD/R+OMT group(0.1,0.2,0.4 mmol·L^(-1)).MTT assay was used to detect cell survival rate.Apoptosis was detected by Hoechst 33342 and Annexin V-FITC.The content of reactive oxygen species(ROS)was detected by flow cytometry.The expressions of p-AMPK,AMPK,p-mTOR,mTOR,Beclin 1,LC3B,p62 andβ-actin were detected by Western blot.Meanwhile,the role of autophagy in the effect of OMT was explored by adding autophagy inhibitor 3-methyladenine(3-MA).Results Compared with CON group,the survival rate of OGD/R group significantly decreased(P<0.01),cell apoptosis rate and intracellular ROS production significantly increased(P<0.01),and the ratio of p-AMPK/AMPK and LC3BⅡ/Ⅰ,and the content of beclin 1 increased(P<0.01).p-mTOR/mTOR ratio and p62 content decreased(P<0.01).Compared with OGD/R group,the cell survival rate in OGD/R+OMT group significantly increased(P<0.01),the cell apoptosis rate decreased(P<0.01),the intracellular ROS production decreased(P<0.01),and the ratio of p-AMPK/AMPK and LC3BⅡ/Ⅰ,and the content of Beclin 1 decreased(P<0.01).p-mTOR/mTOR ratio and p62 content increased(P<0.01).Compared with OGD/R group,the cell survival rate of OGD/R+3-MA group significantly increased(P<0.01),and cell survival rate of OGD/R+3-MA group also had significant difference compared with OGD/R+OMT group and OGD/R+OMT+3-MA group.Conclusions OMT has a protective effect on OGD/R-induced AS injury,which is related to the inhibition of AMPK/mTOR signaling pathway and the amelioration of autophagy.

关 键 词：氧化苦参碱 氧糖剥夺/复糖复氧 星形胶质细胞 自噬 氧化应激 AMPK/mTOR途径 

分 类 号：R284.1[医药卫生—中药学] R322.81[医药卫生—中医学] R329.24R392R852.15